The long term goal of the proposal is to identify and purify the proteins involved in the replication of chloroplast DNA (ctDNA) followed by studying in vitro replication using purified proteins. Replication of ctDNA proceeds by the D-loop mechanism and involves both replicative intermediates produced by Cairn's mode of replication and rolling circle mechanism. We have mapped the origins of replication on the restriction endonuclease map of the pea ctDNA by electron microscopy. We have also developed a crude replication system that contains about 30 polypeptides and shows maximum DNA synthesis with recombinants pCP 12-7 and pCB 1-12 that have been found to contain the two D-loops. We now propose to progressively subclone these ctDNA fragments to eventually obtain the minimal ctDNA sequences that can replicate in an in vitro replication system. The exact sequences of the origins will be identified by obtaining deletion mutants followed by base substitution experiments. The D-loop DNA from supercoiled ctDNA will be analyzed for precisely mapping the initiation sites of replication and for the presence or absence of ribonucleotides as primers. We have obtained a pure preparation of topoisomerase I from the replication complex, and now plan to study its role in initiation of replication. We have obtained four DNA binding proteins whose role(s) in replication will also be studied. Experiments are proposed to find out whether a specific DNA primase or the chloroplast RNA polymerase serves to prime the initiation of replication. The replication of D-loop containing plasmids in vitro will be studied using pure DNA polymerase, topoisomerase I priming enzyme, DNA binding proteins, and any as yet unidentified proteins that are needed in the replication of ctDNA.